A widely used microbial pesticide is derived from the soil microbe Bacillus thuringiensis (Bt). Bt is a gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. The crystal protein, often referred to as the .delta.-endotoxin, has two forms: a nontoxic protoxin with an approximate molecular weight (MW) of 130 kilodaltons (kD); and a toxic form having an approximate MW of 68 kD. The crystalline protein inclusions contain the protoxin protein which is activated in the gut of larvae of a number of insect species. During activation, the protoxin is cleaved, the toxic moiety residing in an amino-proximal 58-68 kD polypeptide. In vivo, the crystal is activated by being solubilized and converted to toxic form by the alkalinity and proteases of the insect gut.
The toxic activity of the protein produced by Bt is highly specific to particular insect species and is recognized as safe to higher vertebrates. Numerous reports have shown that the intrasporal crystal proteins isolated from many strains of Bt possess extremely high levels of toxicity specific for Lepidopteran larvae, or Coleopteran larvae, with an effective concentration required to inhibit 50% of larval growth in the range of 1 ng/ml of diet for the most sensitive insects (MacIntosh et al., J. Invert. Pathol. 565 (1990) 258).
The cloning, sequencing and expression of the Bt protein gene in other bacterial hosts has been described (International Publication No. WO 93/04587, EP Appln. No. 89300388.9, EP Appln. No. 90304996.3, and U.S. Pat. No. 5,286,485). However, expression of insecticidal protein genes derived from Bt in plants has been extremely difficult, and typically, only low levels of protein have been obtained in transgenic plants (Vaeck et al., Nature, 328 (1987) 33; Barton et al., Plant Physiol., 85 (1987) 1103; and Fischoff et al., Bio/Technology, 5 (1987) 807).
One possible explanation for low expression of the native Bt gene in transgenic plants is that the codon usage in a native Bt protein gene is significantly different from that of a typical plant gene (EP Appln. No. 89309069.6). Codon usage may influence the expression of genes at the level of translation, transcription or mRNA processing.
Another possible reason for low levels of expression of the native Bt gene in transgenic plants may be due to fortuitous transcription processing sites which produce aberrant forms of mRNA (International Publication No. WO 93/07278). Possible processing sites include polyadenylation sites, intron splicing sites, transcriptional termination signals and transport signals. Fortuitous occurrence of such processing sites in a coding region may complicate the expression of a gene in a transgenic host.
To optimize an insecticidal gene for expression in plants, attempts have been made to alter the native Bt gene to resemble, as much as possible, genes naturally contained within the host plant to be transformed. For example, U.S. Pat. No. 5,380,831 to Adang et al. describes a chemically synthesized gene encoding an insecticidal protein which is functionally equivalent to a native insecticidal protein of Bt, and which is designed to be expressed in plants at a level higher than a native Bt gene. The synthetic gene is at least approximately 85% homologous to a native insecticidal protein gene of Bt and is designed such that its distribution frequency of codon usage deviates no more than 25% of highly expressed plant genes, and preferably no more than about 10%. The synthetic gene has GC and TA doublet avoidance indices, based on the frequency in a host gene sequence, that deviates from that of the host plant by no more than about 10-15%, and has a GC content of about 45%.
International Publication No. WO 93/07278 describes a synthetic Bt crystal protein gene in which codon usage has been altered in order to increase expression in maize. The synthetic gene is at least approximately 66% homologous to a native insecticidal protein gene of Bt and 98% homologous to a pure maize optimized gene. The synthetic gene has a GC content of from 50-64% and does not have prolines at the 3' end of the sequence.